Mean Fluorescence Intensity Values Research Articles

Determination of the mean duration of recent infection and false recency rate for the HIV triplex multiplex bead assay.

We developed the HIV Triplex multiplex bead assay to identify and serotype HIV infection with high sensitivity and specificity; and distinguish recent from long-term HIV-1 infections. It can facilitate accurate incidence estimation, while reducing the number of tests and blood collected, which is highly desirable for use in future studies and surveys. Using previously collected, treatment-naive longitudinal seroconversion HIV-1 positive panels and specimens from individuals infected for >12 months, we determined the assay's mean duration of recent infection (MDRI) and false-recency rate (FRR) respectively, at various mean fluorescent intensity (MFI) cutoffs. We tested seroconversion specimens (N = 814) from 142 individuals infected with HIV-1 subtypes B, C, or AE, and 1341 cross-sectional specimens from individuals infected >12 months. The MFI cutoffs of 1000 to 2000 were evaluated for recency classification, including an MFI of 1250 corresponding to the limiting antigen avidity enzyme immunoassay (LAg-EIA) cutoff of 1.5 normalized optical density for MDRI and FRR. We used four statistical methods: Methods 1 and 2 used the empirically balanced observation time approach. Method 2 MFI values were raised to power = 1.33, based on a repeated measures model to linearize the relationship between MFI and time points, whereas Method 1 was not linearized. Methods 3 and 4 employed quadratic and linear interpolations for each seroconversion panel. FRR was calculated by dividing the number of specimens misclassified as recent by the total number of specimens tested. MDRI values ranged from 135-146 days at MFI = 1000 to 229-279 days at MFI = 2000 by the 4 methods. FRR varied from 0.15%-1.27% with increasing MFI cutoff. At MFI = 1250, the average MDRI of 4 methods was 169 days and ranged from 159-183 with overlapping 95% CIs and FRR = 0.52%. The HIV Triplex assay demonstrates a longer dynamic range compared to current HIV recency assays with a low FRR for cutoffs examined. With a longer dynamic range and low FRR, the MDRI for recent infection can be extended as appropriate to detect more recent infections, increasing the value of incidence assays benefiting public health surveillance and future surveys.

B-053 Four years summary of flow cytometry live cell based assay for detecting serum anti-aquaporin 4 IgG and anti-myelin oligodendrocyte glycoprotein IgG in a Korean reference laboratory

Abstract Background In the diagnosis of neuromyelitis optica spectrum disorder (NMOSD), it is important to sensitively detect anti-aquaporin 4 (AQP4) IgG and anti-myelin oligodenrocyte glycoprotein (MOG) IgG in patients' serum. NMOSD is a disease that shows 3-7 times more common prevalence in Far East Asia than in the West. According to the IPND diagnostic criteria, it is recommended to diagnose as a cell based assay using full length recombinant protein. In this study, we would like to review 4 years results of AQP4, MOG requested to Eone laboratories in Korea performed with flow cytometry live cell based assay (FC-CBA). Methods FC-CBA was performed using piRES2 cell transfected with AQP4, MOG and stained with DsRed2. Subcultured cells were counted with 10^6, and reacted with patients' serum for 1 hour in a refrigerated state. Thereafter, reaction with FITC anti-human IgG for 2nd antibody for 30 minutes, and 10,000 cells were read from Navius (Beckman Coulter). The mean fluorescence intensity (MFI) ratio , which is the ratio obtained by dividing the MFI value of both FITC and DsRed2 positive cells and DsRed2 positive cells to negative cells, was used to determine positive, borderline, and negative for the serum sample. AQP4 reviewed cases during the period from November 2018 to January 2024, and MOG reviewed cases during the period from December 2019 to January 2024. Indirect immunofluorescence assay using formalin fixed cells is commercially available and compared to the FC-CBA of this institution. Results During the study period, 4,733 cases of AQP4 were performed, and 23,540 cases of MOG were performed by FC-CBA. Among them, AQP4 was positive in 405 cases (8.5%) and borderline in 71 cases (1.5%), and all others were negative. MOG was 2,597 positive (11%), borderline in 1,144 cases (4.8%), and all others were negative. The positive and borderline cutoffs for MFI ratio were changed during the study period, and the positive rate was significantly changed. Conclusions Eone laboratories are the only institution in Korea that performs live cell based assay using flow cytometry for AQP4 and MOG IgG, and receives samples from patients suspected of NMOSD at university hospitals. When reading with MFI ratio, it is important to properly set the cutoff to determine the positivity rate.

A new strategy for systematically classifying HLA alleles into serological specificities: Update and refinement.

HLA antigens were historically defined according to the unique reactivity pattern of cells expressing HLA molecules with distinctive clusters of allo-antisera and/or monoclonal antibodies. Subsequently, amino acid residues determining epitopes (DEP) in the HLA molecule were correlated with reactivity patterns. In current clinical practice, the presence of allo-antibodies is assessed using Luminex-based solid phase single antigen bead (SAB) assays for transplantation. Recently, novel antigens were proposed for HLA molecules with DEP patterns that do not match any serologically defined antigens recognised by the WHO Nomenclature Committee. To validate the antigens, mean fluorescence intensity values of SABs tested on >13,000 patients' sera were extracted from clinical databases and analysed by scatter plots using a linear regression model. We found that when two proteins were considered as the same antigen in the original study, for example, HLA-A*02:01 and -A*02:06, their correlation ranked among the highest values at each locus. In contrast, discrete asymmetric outliers were observed when there were different antigens, for example, HLA-A*30:01 and -A*30:02, allowing validation and confirmation of 20 novel antigens for HLA-A, -B, -C and -DR. The outliers were confirmed to be true or false by flow cytometric crossmatches. In addition to the previously defined residues for antigen assignments, findings suggest that further distinction should be made for common antigens by including the substitutions at residue 67 of HLA-B, 67 and 74 of -DR. These serologic analyses can be applied systematically to identify and confirm novel antigens. These developments will lead to designing optimal SAB panels and further improving virtual donor-specific antibodies assessment.

Deep Immunoprofiling of Large-Scale Tuberculosis Dataset at Single Cell Resolution Reveals a CD81bright γδ T Cell Population Associated with Latency.

Tuberculosis (TB) remains one of the leading causes of death among infectious diseases, with 10.6 million new cases and 1.3 million deaths reported in 2022, according to the most recent WHO report. Early studies have shown an expansion of γδ T cells following TB infection in both experimental models and humans, indicating their abundance among lung lymphocytes and suggesting a role in protective immune responses against Mycobacterium tuberculosis (M. tuberculosis) infection. In this study, we hypothesized that distinct subsets of γδ T cells are associated with either protection against or disease progression in TB. To explore this, we applied large-scale scRNA-seq and bulk RNA-seq data integration to define the phenotypic and molecular characteristics of peripheral blood γδ T cells. Our analysis identified five unique γδ T subclusters, each with distinct functional profiles. Notably, we identified a unique cluster significantly enriched in the TCR signaling pathway, with high CD81 expression as a conserved marker. This distinct molecular signature suggests a specialized role for this cluster in immune signaling and regulation of immune response against M. tuberculosis. Flow cytometry confirmed our in silico results, showing that the mean fluorescence intensity (MFI) values of CD81 expression on γδ T cells were significantly increased in individuals with latent TB infection (TBI) compared to those with active TB (ATB). This finding underscores the importance of CD81 and its associated signaling mechanisms in modulating the activity and function of γδ T cells under TBI conditions, providing insights into potential therapeutic targets for TB management.

Abstract We074: Macrophages Endocytose the Extracellular Matrix Component Hyaluronan

Background: Following myocardial infarction, macrophages clear cellular debris during remodeling of the extracellular matrix (ECM). The presence of macrophages coincides with a significant increase in the ECM component hyaluronan (HA). HA is produced in copious amounts following injury, such as myocardial infarction, and HA may interfere with myocardial wound healing. Because macrophages express hyaluronan receptors and are known to facilitate ECM remodeling, it is possible that macrophages remove HA early after MI. The extent to which macrophages clear HA is not known. Goal: To determine the extent to which macrophages endocytose hyaluronan (HA). Methods and Results: Bone marrow derived macrophages (BMDMs) were polarized into a pro-inflammatory (M1) phenotype using LPS and INF-g, or pro-reparative phenotype (M2) using IL-4 and IL-13. In addition to the polarization factors, cells were treated with 5,000 ng/ml of a fluorescein-labeled HA or a vehicle for 24 hours. At the time of collection, cells were stained with trypan blue to quench surface fluorescence and flow cytometry was used to measure the uptake of labeled HA. An increase in cellular fluorescence intensity after HA treatment indicates the extent of endocytosis of HA. Mean fluorescence intensity (MFI) values were recorded and used to calculate the change in fluorescence between the treated and control groups (DMFI). A significant increase in DMFI was observed when both M1 and M2 macrophages were compared to naïve (M0) controls. Conclusion: Macrophages endocytose HA, and polarized macrophages endocytose HA at a greater level than naïve macrophages. Hence, polarized macrophages may play a role in clearing HA in the heart following injury. We speculate that enhancement of macrophage-mediated clearance of HA may improve acute scar formation following myocardial infarction.

A simple predictor for donor-specific anti-HLA antibody desensitisation in haploidentical haematopoietic stem cell transplantation.

Donor-specific HLA antibody (DSA) has been recognised as an independent risk factor for graft failure in patients undergoing haploidentical haematopoietic stem cell transplantation (HID HSCT). Therapeutic plasma exchange (TPE), as a first-line strategy for DSA desensitisation, can promptly reduce serum DSA levels. This study aimed to investigate DSA characteristics and identify a biomarker predicting the efficacy of DSA desensitisation in patients proceeding to HID HSCT. We retrospectively enrolled 32 patients with DSA from April 2021 to January 2024, and analysed the mean fluorescence intensity (MFI) value of DSA at the different time points of desensitisation treatment. Compared with baseline DSA level before TPE, the median MFI of HLA class I DSA was reduced from 8178.6 to 795.3 (p < 0.001), and HLA class II DSA decreased from 6210.9 to 808.8 (p < 0.001) after TPE. The DSA level in 1:16 diluted pre-TPE serum correlated well with DSA value in post-TPE serum (class I, r = 0.85, p < 0.0001; class II, r = 0.94, p < 0.0001), predicting TPE efficacy in 84.4% of patients. Based on the degree of DSA reduction after TPE, patients were divided into complete responders (decreased by >70%), partial responders (decreased by 30 to 70%) and non-responders (decreased by <30%) and the percentages were 43.8%, 25% and 31.2%, respectively. Non-responders receiving aggressive immunotherapy had longer overall survival compared to those receiving standard strategies (p < 0.05). The 1:16 diluted pre-TPE serum may predict the efficacy of TPE and allow for more rational immunotherapy strategy for patients with DSA proceeding to HID HSCT.

Pseudomonas aeruginosa infection correlates with high MFI donor-specific antibody development following lung transplantation with consequential graft loss and shortened CLAD-free survival

BackgroundDonor-specific antibodies (DSAs) are common following lung transplantation (LuTx), yet their role in graft damage is inconclusive. Mean fluorescent intensity (MFI) is the main read-out of DSA diagnostics; however its value is often disregarded when analyzing unwanted post-transplant outcomes such as graft loss or chronic lung allograft dysfunction (CLAD). Here we aim to evaluate an MFI stratification method in these outcomes.MethodsA cohort of 87 LuTx recipients has been analyzed, in which a cutoff of 8000 MFI has been determined for high MFI based on clinically relevant data. Accordingly, recipients were divided into DSA-negative, DSA-low and DSA-high subgroups. Both graft survival and CLAD-free survival were evaluated. Among factors that may contribute to DSA development we analyzed Pseudomonas aeruginosa (P. aeruginosa) infection in bronchoalveolar lavage (BAL) specimens.ResultsHigh MFI DSAs contributed to clinical antibody-mediated rejection (AMR) and were associated with significantly worse graft (HR: 5.77, p < 0.0001) and CLAD-free survival (HR: 6.47, p = 0.019) compared to low or negative MFI DSA levels. Analysis of BAL specimens revealed a strong correlation between DSA status, P. aeruginosa infection and BAL neutrophilia. DSA-high status and clinical AMR were both independent prognosticators for decreased graft and CLAD-free survival in our multivariate Cox-regression models, whereas BAL neutrophilia was associated with worse graft survival.ConclusionsP. aeruginosa infection rates are elevated in recipients with a strong DSA response. Our results indicate that the simultaneous interpretation of MFI values and BAL neutrophilia is a feasible approach for risk evaluation and may help clinicians when to initiate DSA desensitization therapy, as early intervention could improve prognosis.

Measure of minimal residual burden on circulating tumor cells with over-expression of programmed death-ligand 1 as a dynamic biomarker in patients with colorectal cancer.

e14553 Background: In stage III colorectal cancer (CRC) patients, the extent of oxaliplatin-combination as an adjuvant therapy is still undetermined. Approx. 25-50% Stage II-III CRC patients are known to develop recurrence and metastasis even after comprehensive treatment, attributed to occult disease and Minimal Residual Disease (MRD). Circulating Tumor Cells (CTCs) are bio-mechanistic extravasation source for micro-metastatic disease. CRC patients, with lower adjuvant therapy from 3 to 6 months are known to exhibit increased CTCs and positivity rate due to the emergence of resistant clones. Assays to detect CTCs and expression of Programmed Cell Death-Ligand 1 (PD-L1) as a dynamic biomarker simultaneously has wide clinical implications. Especially, when the tissue biopsy is not inadequate to detect molecular targets for immune checkpoint inhibitor (ICT) therapy. Methods: Retrospectively, 182 CRC cancer patients were analyzed for the presence of CTC and distribution at baseline and follow-ups (up to 0-4 follow ups). The peripheral blood 1.5 ml was analyzed using the OncoDiscover platform approved by CDSCO, India consisting multifunctional magneto-nanosystem mediated by anti-epithelial cellular adhesion molecule (EpCAM) antibody. CTCs were analyzed in patients with early stages- pre and post treatment, progressive disease, Disease Free Status (DFS) and in metastasis. The isolated cells were immune-stained to detect CK-18 +ve, CD45 -ve, DAPI +ve and PD-L1 +ve expression. PD-L1 expression in CTCs was validated by analyzing the linear intensity gradients of the fluorescence signals. CTCs were termed as PD-L1 -ve based on a weak or no detectable fluorescence signal and termed +ve based on a high fluorescence signal using automated image acquisition Zeiss microscope. Results: In a cohort of 182 CRC patient samples, n = 128 (70.3 %) showed the presence of CTCs. The fluorescence intensity for PD-L1 expression as a robust functional assay on CTCs for molecular characterization was developed. The distribution of CTCs ranged from 1-9. The mean fluorescence intensity value and cut-off for the expression of PD-L1 in CTCs was accounted to be ~1.02. Interestingly, in n = 54 (42.2 %) patients, CTCs showed positive expression of PD-L1. CTC +ve patients with PD-L1 expression were associated at all stages and even in early stage, progressive disease, and metastasis. Patients without any CTCs n = 54 (29.7%) either had clinically stable disease or were in DFS with no radio-graphical image. Conclusions: The presence of PD-L1 over-expression on CTCs is a dynamic biomarker in blood, showing the progression of disease even in patients with DFS status. CTC enumeration and assessing the PD-L1 expression on CTCs could offer highly individualized treatment plans in CRC patients.

HER2 expression-independent antitumor effect of trastuzumab deruxtecan (T-DXd) on pediatric solid tumors.

e15016 Background: Treatment of pediatric solid tumors shows limited improvement even with intensification of conventional chemotherapy, suggesting novel approaches such as targeted therapies are needed. Trastuzumab Deruxtecan (T-DXd), a HER2-targeting antibody–drug conjugate, exhibits significant anti-cancer activity in breast cancers with a broad range of HER2 expression. T-DXd might be a promising agent for refractory or relapsed pediatric solid tumors, but its efficacy in pediatric tumors needs to be studied. Methods: Anti-cancer efficacy of T-DXd and its payload DXd were assessed in vitro by utilizing our in-house cancer cell line models. Cytotoxicity assays were performed for 60 pediatric cancer cell lines, including Neuroblastoma (NB) = 25, Ewing sarcoma / Ewing sarcoma family of tumors (EWS / EWSFT) = 17, Rhabdomyosarcoma (RMS) = 10, Others (Osteosarcoma, Brain tumor, Wilms tumor, Hepatoblastoma, and Malignant rhabdoid tumor (MRT) = 8, and 2 breast cancer cell lines expressing HER2 high or low (IHC score 2+ or 0) as positive or low control, respectively. We analyzed HER2 expression on the cell surface by flow cytometry and evaluated relative mean fluorescence intensity (MFI) values. Results: In the flow cytometric assessment, pediatric cancer cell lines showed smaller median MFI of 1.40 (0.67 - 2.71) compared with breast cancer control with HER2 high expression (33.77). The median MFI of each cell lines were 0.94 in NB, 1.76 in EWS / EWSFT, 1.42 in RMS, 1.52 in other pediatric cancers. Although low or no expression of HER2, certain inhibitory activities to the cell growth were observed for T-DXd against a part of NB, EWS, RMS and MRT cell lines, with the minimum IC50 values of 14.5 nM, 6.5 nM, 10.2 nM, 8.1 nM, respectively. In NB, EWS and RMS, a wide range of sensitivity to T-DXd were seen among different cell lines in the same disease. Most of cell lines showed high sensitivity to DXd (Median IC50 value: 0.90 nM [0.08 - 6.46 nM]), but, among them, cell lines with lower sensitivity to DXd tend to show less sensitivity to T-DXd. There was no clear correlation between sensitivity to T-DXd and HER2 expression among pediatric cancer cell lines, indicating that other mechanism such as intrinsic sensitivity to payload might affect antitumor activity of T-DXd. Conclusions: T-DXd exhibited antitumor efficacy against pediatric solid tumors in vitro irrespective of HER2 expression. Our results suggest that T-DXd might be an alternative therapy for pediatric solid tumor cases for whom conventional chemotherapies and other targeting therapies are ineffective. This study supports further investigation for T-DXd in this population.

Effect of electroacupuncture of Governor Vessel on mitochondrial fusion and proliferation and differentiation of neural stem cells in spinal cord injury rats.

To observe the effect of electroacupuncture (EA) at "Dazhui" (GV14) and "Jizhong"(GV6) of the Governor Vessel (GV) on mitochondrial fusion and neural stem cell (NSC) proliferation and differentiation in the spinal cord of rats with spinal cord injury (SCI), so as to investigate its mechanisms underlying improvement of SCI. SD rats were randomly divided into sham operation, model and EA groups, with 15 rats in each group. The SCI model was established by using a precision impactor. EA (20 Hz/100 Hz, 1-2 mA) was applied to GV14 and GV6 for 30 min, once daily for 14 days. The rats' hindlimb locomotor function in each group was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor scale. Histopathological changes of the injured spinal cord tissue and the number of neurons were evaluated after H.E. staining and Nissl staining. The expressions of Nestin, mitochondrial fusion-related protein optic atrophy-1 (OPA1) and NSC markers sex-determining region Y-box 2 (SOX2) in the injured spinal cord tissue were detected by immunofluorescence staining. The protein and mRNA expression levels of Nestin in the spinal cord tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the sham operation group, the BBB scores after modeling, and the number of neurons were significantly decreased (P<0.001), while the mean fluorescence intensity values of Nestin, SOX2 and OPA1, and the expressions of Nestin mRNA and protein considerably increased (P<0.001, P<0.01, P<0.05) in the model group. After EA intervention and in comparison with the model group, the BBB scores at the 7th and 14th day, the number of neurons, the mean fluorescence intensity values of Nestin, SOX2 and OPA1, and the expressions of Nestin mRNA and protein were strikingly increased (P<0.05, P<0.01, P<0.001) in the EA group. H.E. staining showed swollen, ruptured and necrotic neurons of the spinal cord, with a large number of vacuoles and severe inflammatory cell infiltration after modeling, which was relatively milder in the EA group. EA stimulation of GV14 and GV6 can promote the recovery of motor function in rats with SCI, which may be related to its effects in promoting mitochondrial fusion and enhancing the proliferation and differentiation of NSCs.

Role of HLA matching and donor specific antibody development in long-term survival, acute rejection and cardiac allograft vasculopathy

Although there are different data supporting benefits of HLA matching in kidney transplantation, its role in heart transplantation is still unclear. HLA mismatch (MM) between donor and recipient can lead to the development of donor-specific antibodies (DSA) which produces negative events on the outcome of heart transplantation. Moreover, DSAs are involved in the development of antibody-mediated rejection (AMR) and are associated with an increase in cardiac allograft vasculopathy (CAV). In this study it is analyzed retrospectively the influence of HLA matching and anti-HLA antibodies on overall survival, AMR and CAV in heart transplantation. For this retrospective study are recruited heart transplanted patients at the Cardiac Transplantation Centre of Naples between 2000 and 2019. Among the 155 heart transplant patients, the mean number of HLA-A, B, -DR MM (0 to 6) between donor and recipient was 4.5 ± 1.1. The results show a negative association between MM HLA-DR and survival (p = 0.01). Comparison of patients with 0–1 MM at each locus to all others with 2 MM, for both HLA class I and class II, has not showed significant differences in the development of CAV. Our analysis detected DSA in 38.1% of patients. The production of de novo DSA reveals that there is not an influence on survival (p = 0.72) and/or AMR (p = 0.39). Instead, there is an association between the production of DSA class II and the probability of CAV development (p = 0.03). Mean fluorescence intensity (MFI) values were significantly higher in CAV-positive patients that CAV-negative patients (p = 0.02). Prospective studies are needed to evaluate HLA class II matching as an additional parameter for heart allocation, especially considering the increment of waiting list time.

Radiation exposure lymphocyte damage assessed by γ-H2AX level using flow cytometry

DNA double-strand breaks (DSBs) are considered the most relevant lesions to the DNA damage of ionizing radiation (IR), and γ-H2AX foci in peripheral blood lymphocytes are regarded as an adequate marker for DSB quantitative studies. This study aimed to investigate IR-induced DNA damage in mice through γ-H2AX fluorescence analyses by flow cytometry (FCM). The levels of γ-H2AX in CD4/CD8/B220-positive lymphocytes were quantified by FCM through mean fluorescence intensity (MFI) values. Peripheral venous blood samples were collected for evaluation, and all the control groups were restrained from irradiation. For external irradiation experiments, the dose-dependency of MFI values and temporal alternations were assessed both in vitro and in vivo. External radiation exposure damage was positively correlated with the absorbed radiation dose, and the lymphocyte recovered from damage within 3 days. I-131 sodium iodide solution (74 MBq) was injected into the mice intraperitoneally for internal irradiation experiments. Gamma counting and γH2AX foci analyses were performed at 1 h and 24 h by the group. The blood-to-blood S values (Sblood←blood) were applied for the blood-absorbed dose estimation. Internal low-dose-irradiation-induced damage was proved to recover within 24 h. The FCM method was found to be an effective way of quantitatively assessing IR-induced DNA damage.

Predicting flow cytometry crossmatch results from single-antigen bead testing.

The aim of this study was to devise an algorithm that would predict flow cytometry crossmatch (FCXM) results using single-antigen bead (SAB) mean fluorescent intensity (MFI) levels using samples received through the National External Quality Assurance Scheme (NEQAS) 2B external proficiency testing scheme between 2019 and 2023. A total of 159 serum samples were retrospectively screened using LABScreen Single Antigen Class I and II (SAB), and 40 peripheral blood samples were human leucocyte antigen (HLA) typed with LABType SSO. Donor-specific antibodies were identified for each cell-serum combination tested, and cumulative MFI values were calculated for each test before correlating the screening result with the consensus crossmatch results for this scheme. HLA Class I MFIs were combined to predict the T cell crossmatch. For the B cell crossmatch prediction, two options were considered: (i) HLA Class II MFI values alone and (ii) HLA Class I+Class II MFIs. Receiver operating characteristic analysis was carried out to identify the combined MFI threshold that predicted NEQAS consensus results with the greatest sensitivity and specificity. HLA Class I combined MFI >5000 predicted T cell crossmatch results with 96% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 92% negative predictive value (NPV). For B cell results, HLA Class I+Class II combined MFIs >11,000 gave the best model, showing 97% sensitivity, 82% specificity, 96% PPV and 85% NPV. However, for samples with only HLA Class II sensitization, combined MFIs >13,000 improved the B cell crossmatch predictions: 92% sensitivity, 95% specificity, 96% PPV and 91% NPV. Using this model, combined MFI can be used to predict the immunological risk posed by donor-specific antibodies when it is not possible to carry out an FCXM.

Detection of effusion tumor cells under different storage and processing conditions.

Circulating tumor cells (CTCs) shed into blood provide prognostic and/or predictive information. Previously, the authors established an assay to detect carcinoma cells from pleural fluid, termed effusion tumor cells (ETCs), by employing an immunofluorescence-based CTC-identification platform (RareCyte) on air-dried unstained ThinPrep (TP) slides. To facilitate clinical integration, they evaluated different slide processing and storage conditions, hypothesizing that alternative comparable conditions for ETC detection exist. The authors enumerated ETCs on RareCyte, using morphology and mean fluorescence intensity (MFI) cutoffs of >100 arbitrary units (a.u.) for epithelial cellular adhesion molecule (EpCAM) and <100 a.u. for CD45. They analyzed malignant pleural fluid from three patients under seven processing and/or staining conditions, three patients after short-term storage under three conditions, and seven samples following long-term storage at -80°C. MFI values of 4',6-diamidino-2-phenylindol, cytokeratin, CD45, and EpCAM were compared. ETCs were detected in all conditions. Among the different processing conditions tested, the ethanol-fixed, unstained TP was most similar to the previously established air-dried, unstained TP protocol. All smears and Pap-stained TPs had significantly different marker MFIs from the established condition. After short-term storage, the established condition showed comparable results, but ethanol-fixed and Pap-stained slides showed significant differences. ETCs were detectable after long-term storage at -80°C in comparable numbers to freshly prepared slides, but most marker MFIs were significantly different. It is possible to detect ETCs under different processing and storage conditions, lending promise to the application of this method in broader settings. Because of decreased immunofluorescence-signature distinctions between cells, morphology may need to play a larger role.

Establish a Graded Method to Avoid HLA Class I Antibodies Corresponding Antigen and Combining HLAMatchmaker Application in Improving the CCI Value after Platelet Transfusion for Patients with IPTR

To establish a graded method to avoid mean fluorescence intensity (MFI) threshold of HLA Class I antibodies corresponding antigen, and the HLAMatchmaker program has been used to select the minimum mismatch value of donor-patient epitopes. Evaluate the application value of combining both methods in selecting HLA compatible platelets (PTL) for patients with immune platelet transfusion failure (IPTR) in improving platelet the corrected count increment (CCI). A total 7 807 PLT cross-matching compatible were performed by the solid-phase red cell adherence (SPRCA) method for 51 IPTR patients. The Luminex single antigen flow cytometry was used to detect HLA Class I antibodies in patients, and detected the MFI value for different specificity antigens of HLA Class I antibodies, was graded into strong positive group (MFI>4 000, level 1), medium positive group (1 000< MFI≤4 000, 2), weak positive group (500< MFI≤1 000, 3), and one negative control group (MFI≤500). The results of 7 807 SPRCA their negative/positive reaction wells were enrolled and statistically analyzed in different grades and the four groups, the statistical differences between the four groups were compared. Multiple applications for the select HLA Class I compatible donor events were made for patients in two cases, and HLAMatchmaker program was used to calculate the number of HLA Class I epitopes mismatches between the donors and patients. The donor with the minimum number of epitopes mismatches was selected, while avoiding the corresponding antigens of HLA Class I antibodies in levels 1 and 2, the provision of HLA compatible platelets for IPTR. After the transfusions, the CCI value of the platelet transfusion efficacy evaluation index was calculated, and the clinical evaluation of the transfusion effect was obtained through statistical analysis. There were statistically significant differences in the positive results of SPRCA immunoassay among the strong positive group, medium positive group, and weak positive group of 51 IPTR patients with different specific of HLA -I class antibodies and corresponding antigens(all P <0.001). The positive results showed a range from high to low, with strong positive group>medium positive group>weak positive group. There were a statistical difference among between the strongly positive or moderately positive groups and the negative control group(P <0.001). There was no statistical difference between the weakly positive group and the negative control group(P >0.05). The strong positive group was set as the corresponding specific HLA Class I site corresponding antigen grade 1 avoidance threshold, the medium positive group as the grade 2 avoidance thresholds, and the weak positive group as the grade 3 avoidance threshold. In the case of donor platelet shortage, it is not necessary to avoid the weak positive group. Avoiding the strategy of donor antigens and HLAMatchmaker program scores ≤7 corresponding to HLA Class I antibodies of levels 1 and 2, with CCI values>4.5×109/L within 24 hours, can obtain effective clinical platelet transfusion conclusions. When selecting HLA Class I compatible donors for IPTR patients, the grading avoids HLA Class I antibodies corresponding to donor antigens, and the donor selection strategy with the minimum scores of HLAMatchmaker program is comprehensively selected. The negative result confirmed by platelet cross-matching experiments has certain practical application value for improving platelet count in IPTR patients.

Diagnosis of chronic B-cell lymphoproliferative disease in peripheral blood=how machine learning may help to the interpretation of flow cytometry data.

Flow cytometry (FCM) has become a method of choice for immunologic characterization of chronic lymphoproliferative disease (CLPD). To reduce the potential subjectivities of FCM data interpretation, we developed a machine learning random forest algorithm (RF) allowing unsupervised analysis. This assay relies on 16 parameters obtained from our FCM screening panel, routinely used in the exploration of peripheral blood (PB) samples (mean fluorescence intensity values (MFI) of CD19, CD45, CD5, CD20, CD200, CD23, HLA-DR, CD10 in CD19-gated B cells, ratio of kappa/Lambda, and different ratios of MFI B-cells/T-cells [CD20, CD200, CD23]). The RF algorithm was trained and validated on a large cohort of more than 300 annotated different CLPD cases (chronic B-cell leukemia, mantle cell lymphoma, marginal zone lymphoma, follicular lymphoma, splenic red pulp lymphoma, hairy cell leukemia) and non-tumoral selected from PB samples. The RF algorithm was able to differentiate tumoral from non-tumoral B-cells in all cases and to propose a correct CLPD classification in more than 90% of cases. In conclusion the RF algorithm could be proposed as an interesting help to FCM data interpretation allowing a first B-cells CLPD diagnostic hypothesis and/or to guide the management of complementary analysis (additional immunologic markers and genetic).

The Challenge of Preoperative Panel Reactive Antibody Positivity in Parathyroid Transplantation.

Identifying suitable recipient criteria and matching recipients with appropriate donors are required to increase survival for parathyroid transplant. This study was undertaken to evaluate transplant survival rates while comparing preoperative panel reactive antibody positivity. The study included 14 hypoparathyroidism patients who presented to our clinic for parathyroid transplant. Preoperative ABO compatibility and negative cross-match tests were prioritized for recipient-donor matching, and panel reactive antibody screening tests were performed. During the 24-month follow-up, we evaluated medication use and serum calcium, phosphorus, and parathormone levels of patients. Preoperative panel reactive antibody positivity was assessed in 3 groups. The HLA class I-positive group (mean fluorescence intensity range, 179-1770) showed decreased medication use and stability in serum calcium levels. The HLA class IIpositive (mean fluorescence intensity range, 85-3959) showed decreased medication use by 25% to 50% and returned to their former prescription doses after 12 months. An opposite pattern was observed in 2 patients with panel reactive antibody positivity for both HLA classes (mean fluorescence intensity range, 462-2289), with 1 patient requiring medication for continuing symptoms and the other patient occasionally taking additional magnesium supplementation, despite decreased medication doses after 12 months. Serum calcium levels remained normal, and parathormone and phosphorus levels were elevated. Improving patient symptoms and having no requirement for intravenous calcium replacement are priorities, and monitoring serum levels is the next important step. Varied panel reactive antibody positivities and survival rates indicate a requirement, and each HLA class could require a proper limitation for the mean fluorescence intensity. Preoperative mean fluorescence intensity cut-off value should be <900. Higher mean fluorescence intensity values in panel reactive antibody screenings could increase risk of short-term graft survival after parathyroid transplant. Further studies should include immunological risk assessments by individualizing the outcome with donor-specific antibodies.

Evaluation of different microbead volumes in a single antigen bead assay for HLA antibody detection

Abstract OBJECTIVE S: The multiplexed single antigen bead (SAB) assay is now commonly used for detection of antibodies to human leukocyte antigens (HLA) in the management of solid organ transplantation. Serum is incubated with microbeads individually coated with unique HLA antigens and the presence of antibody is detected using a secondary antibody. Due to its cost, laboratories may use a lower-than-recommended volume of reagent microbeads, although the impact of microbead volume on assay performance has not been reported. The present study seeks to determine the concordance of an SAB assay when different microbead volumes were used. Although this is also a required laboratory practice when modifications are made to an FDA-cleared assay, requirements for the validation of a multiplexed assay are currently not well-standardized. Hence, this study may also serve as an example framework for validating a clinical multiplexed assay. METHODS SAB (LabScreen, One Lambda) was performed on 14 patient specimens [Mei San T1] selected to provide a range of class I and II HLA antibody levels. Each specimen was tested using three microbead volumes of 5 μL (manufacturer-recommended), 3 μL and 2.5 μL. All reactions were tested in a single session to minimize sources of variability. A mean fluorescence intensity (MFI) of ≥ 2000 was used to define positive beads, which is also the institutional threshold for defining incompatible antigens in solid organ transplantation. Concordance between different bead volumes was calculated using Cohen’s Kappa. Bland-Altman plots were used to visualize bias. All analyses were performed at individual bead level. RESULTS Kappa values were: 0.91 (class I) and 0.95 (class II) for 3 μL vs. 5 μL; 0.90 (class I) and 0.93 (class II) for 2.5 μL vs. 5 µL; 0.97 (class I) and 0.93 (class II) for 2.5 μL vs. 3 μL. Positive bias in MFI values was observed with lower bead volumes and was most pronounced in the 2.5 μL vs. 5 μL comparison; in contrast, MFI values between 2.5 µL and 3 µL were more similar. CONCLUSION Lower-than-recommended microbead volume may be used in the SAB assay without significant impact on classification of incompatible antigens. Although positive bias in MFI values was observed with lower microbead volumes, the impact on positive vs. negative beads assignment was minimal. Since the tested microbead volumes represented 12.5-20% of the total reaction volume, such a change could have caused a matrix effect and impacted antibody-bead binding, leading to differences in MFI values. Hence, individual laboratories modifying the SAB assay should validate such modifications before implementation to ensure the accuracy of HLA antibody assignment.

Impact of Immunosuppression Intensified Conditioning Regimen for Patients with Strong Positive Pre-Transplantation Donor Specific Anti-HLA Antibodies (DSAs) Undergoing Haploidentical Hematopoietic Stem Cell Transplantation

Objective: Immunosuppression intensified conditioning regimen has been applied for 10 patients with strong positive pre-transplantation Donor specific Anti-HLA Antibodies (DSAs) undergoing Haploidentical Hematopoietic Stem Cell Transplantation (HSCT), its effects on engraftment of hematopoietic stem cell were examined. Methods: Characteristics of 10 consecutive patients with strong positive pre-transplantation DSAs were retrospectively analyzed, including gender, age, diagnosis, disease status pre-transplantation, presence of DSA, conditioning regimens, engraftment, graft versus host disease (GVHD) incidence and survival. Results: Ten patients [3 males vs 7 females, median age 53.5(36-64)] received immunosuppression intensified conditioning regimen: Fludarabine plus busulfan (FluBu) combining total body irradiation and cyclophosphamide (TBI-Cy). DSA levels gradually decreased in all patients after preparative conditioning, and the mean fluorescence intensity (MFI) values dropped significantly (P＜0.003). The average MFI values dropped 40% at day 7 post-HSCT, 8 cases acquired hematopoietic reconstitution, the median time of neutrophil engraftment was 15.6(10-16) days, and 19(13-22) days for platelet engraftment. One case had primary graft failure and another one had poor graft function. After a median follow-up of 12.5(1.5-27) months, 7 cases remained in disease remission state with negative DSA assay, including 2 cases with MFI values higher than 20000 pre-transplantation. Conclusions：Immunosuppression intensified conditioning regimen can efficiently decrease the Donor specific Anti-HLA Antibodies (DSA) level, which leads to lowered chances of primary graft failure.

Mass Spectrometry Based Detection of Naturally Expressed FVIII in Liver Sinusoidal-like Endothelial Cells from Healthy Donor and HA Patients with Nonsense Mutations

MS Background: Liver sinusoidal endothelial cells (LSEC) are unique endothelial cells and secretion and expression of FVIII is one of their key functions. Pluripotent stem cells from healthy donors can be a source of generating organ-specific endothelial cells in vitro. However, maintaining the specification of LSEC markers remains a challenge. Currently, EBs with a combination of AMP activation,TGF-ß inhibition,and hypoxia are a state of the art to derive LSEC-like EC from IPS. A 2D monolayer-based approach shows potential in culturing these ECs and in maintaining their correct specification (Fig. 1).IPSc-differentiated LSEC were analyzed for the presence of LSEC-specific markers FCGR2b, Stab2 &amp; F8 by rtPCR. Our data shows that day 10 angioblasts mark an increase of venous markers COUP-TF and NT5E under low VEGF-A conditions. Upon LSEC induction LSEC markers are upregulated compared to HUVEC:FCGR2b(2x), Stab2(37x), and F8(7x) (not shown). Flow cytometry(FC) analyses depict a KDR +CD56 + phenotype for mesoderm on day 4 ,a venous CD31 +CD34 +CD184 lowCD73 high phenotype for day 6 angioblast and a CD34 +FCGR2b +Stab2 +LYVE1 + LSEC-LC population successfully maintained until Day 15 (not shown).ELISA measurements (Fig. 2) using a FVIII-A3 specific biotinylated nanobody detects FVIII in LSEC-LC from healthy donor, HA-patient with I22I and in both high risk inhibitor nonsense mutations R1960X and R2228X. These preliminary results warranted the confirmation of detection as well as the need to decipher the differences in the FVIII protein among the healthy donor and patients with HA. Aim: Detection of endogenous FVIII protein in healthy donors and patients using immunofluorescence(IF). Detection of FVIII protein using mass spectrometry (MS) Methods: IF was conducted using a monoclonal antibody (GMA8021) and the difference in protein levels was measured using the mean fluorescence intensity (MFI) values. An immunoprecipitation-based method to detect the natural FVIII protein using MS was developed. Initially, an ultracentrifugation step eliminated the background proteome which allowed for the antibody (SAF8C) based capture of FVIII using a column-based elution technique. The eluted sample was trypsinized and detected via nano-LC coupled to LC/MS Orbitrap. Results: The MFI of the healthy donor is 2.8x, 2.5x, 2.31x, 2x and 1.3x higher than F8KO, HUVEC, HA-patient samples R2228X, R1960X and I22I,respectively. Healthy donor shows the presence of 5 FVIII peptides. Patient sample with mutation I22I shows the presence of 1 FVIII peptide. Patient sample with nonsense mutation R1960X showed 2 FVIII peptides while patient sample with R2228X was unable to show any FVIII peptides. Conclusion: While FVIII detection in multiple overexpressed models of HA have been established, our current LSEC model is a milestone in detecting the natural, native state of the FVIII protein. This LSEC-LC model provides a 7-fold higher F8 expression compared to HUVEC in vitro system. FC analysis shows the right phenotype for modeling IPSc into LSEC. The additional presence of LSEC-specific receptors and confirmation of FVIII in the healthy donor and patients with nonsense mutations enables a more native and precise disease modeling for HA research and therapy.